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Use of genome sequencing to investigate the molecular basis of bacteriaphage interaction of the Escherichia coli O157 typing phages and the elucidation of the biological and public health significance of phage type

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Abstract

Background
Shiga toxin producing Escherichia coli (STEC) O157 causes severe gastrointestinal
disease and haemolytic uremic syndrome, and has a major impact on public health
worldwide with regular outbreaks and sporadic infection. Phage typing, i.e. the
susceptibility of STEC O157 strains to a bank of 16 bacteriophages, has been used in the UK to differentiate STEC O157 for the past 25 years and the phage type (PT)
can be an epidemiological marker of strains associated with severe disease or
associated with cases that occur from foreign travel. However, little is known
about the molecular interactions between the typing phages (TP) and STEC O157.
The aims of this thesis were to use whole genome sequencing to elucidate the
genetic basis for phage typing of STEC O157 and through this understand genetic
differences between strains relevant to disease severity and epidemiology.
Results
Sequencing the STEC O157 TPs revealed that they were clustered into 4 groups
based on sequence similarity that corresponded with their infectivity. Long read
sequencing revealed microevolutionary events occuring in STEC O157 genomes
over a short time period (approximately 1 year), evidenced by the loss and gain of
prophage regions and plasmids. An IncHI2 plasmid was found responsible for a
change in Phage Type (PT) from PT8 to PT54 during two related outbreaks at the
same restaurant. These changes resulted in a strain (PT54) that was fitter under
certain growth conditions and associated with a much larger outbreak (140 as
opposed to 4 cases). TraDIS (Transposon directed Insertion site sequencing) was
used to identify 114 genes associated with phage sensitivity and 44 genes involved
in phage resistance, emphasising the complex nature of identifying specific
genetic markers of phage susceptibility or resistance. Further work is required to
prove their phage-related functions but several are likely to encode novel phage
receptors. Deletion of a Stx2a prophage from a PT21/28 strain led to a strain that
typed as PT32, supporting the concept that the highly pathogenic PT21/28 lineage
I strains emerged from Stx2c+ PT32 strains in the last two decades by acquisition
of Stx2a-encoding prophages.
Conclusions
This body of work has highlighted the complexity of bacteriophage interaction and
investigated the genetic basis for susceptibility and resistance in E. coli. The
grouping of the TPs showed that resistance or susceptibility to all members of a
typing group was likely to be caused by one mechanism. IncHI2 was identified as
one of the markers for the PT54 phenotype. The Stx2a prophage region was
associated with the switch from PT32 to PT21/28, although PT32 strains
containing both Stx2a and Stx2c-encoding prophages have been isolated and can
provide insights into phage variation underpinning the susceptibility to the
relevant typing phages. The TraDIS results indicated that susceptibility or
resistance was governed by multiple genetic factors and not controlled by a single
gene. The significance of LPS for initial protection from phage adsorption was
evident and a number of novel genes controlling phage susceptibility and
resistance identified including the Sap operon and stringent starvation protein A
respectively. While SNP-based typing provides an excellent indication of the
evolution and relatedness of strains, phage typing can provide real insights into
short term evolution of the bacteria as PTs can be altered by mobile elements
such as prophages and plasmids. This study has shown that, although complex,
genetic determinants for PT can be mined from the genome and allow us to
understand the evolution of this zoonotic pathogen between host species and
during outbreaks.